Liquid scintillation counting is a method for determining the amount of one or more radioactive substances. The method is used mainly for determining beta-emitting isotopes, such as .sup.3 H, .sup.14 C and .sup.32 P.
Beta-emitting isotopes decay by emitting energy in the form of a fast electron and a neutrino. The energy liberated in the decay is always constant for a certain radioisotope, but is divided between the electron and the neutrino according to a distribution law. The neutrino can not be detected by using liquid scintillation counting but the electron will through collisional impact transfer some of its energy to the liquid solvent molecules which are then ionized or excited to higher energy levels. Provided that the solvent molecules are mainly of aromatic character and that certain fluorizing compounds are dissolved in the solution, part of the excitation energy deposited by the electron may be transformed to light which can be detected by a photosensitive device such as a photomultiplier. The intensity of the light pulse caused by a decay is proportional to the energy of the electron when ejected from the nucleus. The height of the electrical pulse measured at the output of the photomultiplier device is again proportional to the number of photons in the light pulse. As each decay produces one distinct pulse, with a height proportional to the energy of the beta electron, a certain pulse height distribution, or spectrum, can be recorded. The shape of this pulse distribution not only depends on the decay characteristics but also on the efficiency of the liquid to transform excitation energy into light and the efficiency of the detector to transform photons into detectable electrical pulses. FIG. 1 shows typical pulse height distributions for .sup.3 H and .sup.14 C, measured in a liquid scintillation counter having a logarithmic pulse height scale. The number of pulses in the pulse height distribution detected per time unit is called the count rate.
Quenching of the scintillation light pulse means that the number of photons produced in a decay, where the electron has a certain energy, is diminished. Hence, quenching results generally in both lower pulse heights and lower count rates. As the object in most measurements is to determine the activity, which is equal to the disintegration rate, and not only the count rate, the relation between activity and count rate must be known. This relation is equal to the counting efficiency of the sample. As the counting efficiency may vary from sample to sample even within one measurement batch, it becomes necessary to determine the counting efficiency for each sample. All methods in commercial use depend on the determination of some feature describing the movement of the pulse height distribution with the quench level. In these methods, either the pulse height distribution produced by the sample isotope or by an external gamma radiating source ("external standard") may be used. In any case, determination of the efficiency of an unknown sample relies on calibration of the instrument. This step includes the measurement of a number of calibration samples containing known amounts of the pure radioisotopes under study and having different levels of quench. For each radioisotope, one such quench calibration set must include at least two calibration samples. Each quench set thus results in a quench calibration function, giving counting efficiency as a function of some indication of quench level. In the case of two calibration samples for each radioisotope, the quench function will be a straight line. The quench function provides means to interpolate between and to some extent extrapolate from the calibration points.
As one unknown sample may contain two or more different radioisotopes, the counter must have means for distinguishing between the contribution of each radioisotope and determining their activities. One such multilabeled sample may further have a quench level not equal to any of the calibration samples. Since the spectra of the radioisotopes overlap one another more or less (see FIG. 1), a complicated situation arises for which there are a few solutions available in commercial instruments.
A traditional solution, which will be referred to as the "preset window" method, depends on using the same number of preset pulse height windows with fixed limits as there are radioisotopes in the sample. Thus, in the case of a dual-labeled sample, two windows are used. As an example, in FIG. 1 the limits A and B together define a first counting window, while limits B and C define a second window. Counts falling between limits A and B are hence referred to as "window 1 counts". Using equal number of windows as there are radioisotopes provides for a simple mathematical relation between the count rate in each window, the efficiency of each radioisotope in each window and the activity of each radioisotope. For instance, if two radioisotopes, Q and P, are present in the sample, then the following equations are valid EQU Y.sub.1 =E.sub.Q1 *A.sub.Q +E.sub.P1 *A.sub.P EQU Y.sub.2 =E.sub.Q2 *A.sub.Q +E.sub.P2 *A.sub.P ( 1)
where Y.sub.1 and Y.sub.2 are the measured count rates in window 1 and 2, respectively, and E.sub.Q1, E.sub.P1, E.sub.Q2 and E.sub.P2 are the known counting efficiencies of radioisotopes Q and P in window 1 and window 2, respectively. A.sub.Q and A.sub.P are the unknown activities of the two isotopes. The two equations (1) have two unknowns and can hence be solved by using linear algebra provided that the counting efficiencies E.sub.Q1, E.sub.P1, E.sub.Q2 and E.sub.P2 are known. These counting efficiencies can be calculated by interpolation between or extrapolation from the calibration sample points, which have to be measured and stored prior to counting unknown samples. This is done in the aforementioned step of calibration. In calibration for multi-labeled samples, counting efficiencies and quench level values have to be stored for each radioisotope and each window. Generally, when using preset windows and dual-labeled samples, four quench functions have to be stored in memory.
The main limitation to the preset window method is that the window limits have to be selected so as to suit the quench level of the unknown samples. If the samples have very varying quench levels, the selected windows may be optimal for only some of the samples. Further, the optimal window limits also depend on the relative amounts of the isotopes present, but in general, the relative amounts are not known a priori.